Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, causes significant morbidity and mortality in immunosuppressed individuals, and has been proposed as a risk factor in vascular disease. This virus has evolved multiple mechanisms to usurp cellular signaling and regulatory pathways to facilitate its replication. A key question has been how the virus activates the cell to a state that is optimal for DNA replication, and at the same time alters the levels and activity of selected cellular proteins so that viral replication proceeds at the expense of the host. We have found that one mechanism by which HCMV exploits the host cell involves the ubiquitin-proteasome degradation pathway. A specific target of the virus is the multisubunit Anaphase Promoting Complex (APC) E3 ubiquitin ligase. During the early phase of the infection, this complex is disabled, allowing stabilization and accumulation of its substrates. The effects on the APC include degradation of the APC4 and APC5 subunits, hyperphosphorylation of Cdh1, a loss of binding of both the APC1 subunit and Cdh1 to subcomplex core of the APC (APC subunits 3, 6, 7, 8) and relocalization of the APC subunits. We propose to determine the viral gene(s) that are responsible for this dysregulation of the APC and have developed a high throughput cellular assay for this purpose. There are two complementary approaches that we will take. The first utilizes a library of cDNA constructs corresponding to all of the HCMV open reading frames (ORFs), and the second utilizes a library of HCMV Bacterial Artificial Chromosomes (BACs) that have mutations in each of the ORFs. These results will provide the basis for more in-depth studies on the molecular and cellular mechanisms involved and the functions of these genes with respect to viral replication and pathogenesis. PUBLIC HEALTH RELEVANCE: Human Cytomegalovirus (HCMV) is the major viral cause of birth defects, poses a serious problem for immunocompromised individuals, and has been proposed to be a risk factor for vascular disease. The serious problems associated with HCMV infections have provided a major impetus for understanding the molecular and cellular biology of the virus and the regulatory pathways governing its replication and interactions with the host.